The production system of heterogeneous proteins using recombinant E. coli ensures rapid cellular growth rates and high density cultures using inexpensive substrates and uses relatively well-identified genes in comparison with cases using other organisms, thereby making it possible to design various vector systems for facilitating the high expression and purification of heterogeneous proteins (Jeffrey G. T. and Amanda A. et al, (1997), Applied Biochemistry and Biotechnology 66, 197-238).
However, when E. coli is used as a host cell for the production of eukaryotic proteins, E. coli cannot perform post-translational modification such as glycosylation because it does not possess intracellular factors required for protein maturation. In addition, when a heterogeneous protein is expressed in high levels, it is often accumulated in the form of inclusion bodies, which are insoluble precipitates.
Inclusion bodies are typically formed by interaction between hydrophobic surfaces of folding intermediates of a target protein due to imbalance between the production rate and the folding rate of the target protein. In this case, inclusion bodies may be easily isolated, be typically less affected by proteinases and be accumulated in high concentrations in cells, thereby securing high yields and easy isolation of a target protein. Due to these advantages, the strategy of expressing a protein as inclusion bodies is utilized in the production of proteins unfavorable for in vivo folding. However, a target protein expressed as inclusion bodies requires an additional refolding process to recover its biological activity. The refolding of a target protein to an active form is dependent on experience, and is thus always not successful and makes it difficult to scale up the production of recombinant proteins in industrial scales. In addition, high molecular weight antibody proteins, tissue plasminogen activator (tPA) and factor VIII are very difficult to produce in active forms by a refolding process.
As described above, since inclusion body proteins should be refolded to have their structure and biological activity intact (Andrew D. Guise, Shauna M. West, and Julian B. Chaudhuri (1996), Molecular Biotechnology 6, 53-64), a target protein is expressed as a soluble protein using the so-called “in vivo protein folding technique” to induce its correct three-dimensional structure formation in vivo. Since this technique improves problems caused when a heterogeneous protein is expressed as inclusion bodies, it has an industrial importance in producing heterogeneous proteins in E. coli. 
The following three strategies are typically used for in vivo folding of proteins.
The first strategy involves the control of protein expression sites and culture environments. When a target protein is designed to be expressed in the cytoplasm, although the target protein is harmful to cells, the cells are not damaged, and the protein is mostly expressed in very high levels. Also, this method facilities the preparation of expression vectors. As another method, the secretion of a target protein to the periplasm has advantages of simplifying protein purification and, compared to the method of expressing a protein in the cytoplasm, reducing protein degradation by proteinases and making disulfide bonding possible to some degree due to a relatively oxidative environment. The advantages further include that an authentic protein can be obtained by removing an N-terminal secretory signal. However, a secreted protein may be aggregated, resulting in formation of inclusion bodies, and reduced folding may occur. In a further method, the secretion of a target protein to culture media may solve the problems associated with protein folding and degradation by proteinases. However, E. coli rarely secretes proteins to culture media, and, even when proteins are secreted to media, proteins are greatly diluted, thus making purification rather difficult. This method is effective only in particular proteins and is thus not a generalized method to prevent inclusion bodies from being formed. Also, the fermentation control is frequently used to increase a soluble protein, and, in most cases, is the most economical method (Korean Pat. Application No. 1997-50023). The reduction of culture temperature is not applied to all proteins, but is a very effective method in many cases because it typically leads to decrease the production rate of a protein below the folding rate of the protein, resulting in no accumulation of folding intermediates with strong aggregation to each other (Schein, C. H. and M. H. M. Noteborn (1988), Biotechnology 6, 291-294; More, J. T., Uppal, F. Maley and G. F. Maley (1993), Protein. Expr. Purif. 4, 160-163).
The second strategy involves the co-expression of chaperones and protein foldases. The chaperones refer to proteins that function to help formation of the desired three-dimensional structure of protein and prevent unnecessary intermolecular or intramolecular interactions. Chaperone proteins derived from E. coli include GroEL, GroES, DnaK, HtpG, SecB and PapD, which protect folding intermediates and prevent aggregation and precipitation, and all of the E. coli chaperone proteins except for PapD (present in the periplasmic membrane) are present in the cytoplasm (Korean Pat. Application No. 2003-7008657; Hartl, F. U., R. Holdan and T. Langer (1994), Trends Biochem. Sci. 19, 20-25; Bernadea-Clark, E. and G. Georgiou (1994), American Chem. Soc. Symp. Ser. Vol. 470, ACS). Foldases refer to an auxiliary protein family that serves to facilitate covalent boding or isomerization during folding. Enzymes stimulating the disulfide bond formation of proteins include DsbA, DsbB, DsbC and DsbD (Creighton, T. E., A. Zapun and N. J. Darby (1995), TIBTECH. 13, 18-27; Gottesman, M. E. and W. A. Hendrickson (2000. Curr. Opin. Microbiol. 3, 197-202).
The third strategy involves the use of fusion proteins. Many proteins have been developed as fusion proteins, which include glutathione-S-transferase, maltose-binding protein, Protein A, tumor necrosis factor-α and lysyl-tRNA synthetase (Smith, D. B. and Johnson, K. S. (1988), Gene 67, 31-40.; Bedouelle, H. and Duplay, P. (1988), Euro. J. Biochem. 171, 541-549.; Nisson, B. et al. (1987), Prot. Eng. 1, 107-113; Korean Pat. Application No. 1996-44010). Also, as described in U.S. Pat. No. 6,027,888, a soluble eukaryotic protein having disulfide bonds can be produced by being expressed in a fused form with disulfide isomerase. In addition, as described in Korean Pat. Application No. 2002-0040497, an H-chain human ferritin protein can be produced as a soluble fusion protein with a L-chain human ferritin protein that is expressed in an insoluble form in E. coli. As described above, various attempts were made to express heterogeneous proteins in soluble fusion protein forms. However, the fusion effect varies according to the type of fusion proteins, as follows: fusion proteins are expressed as inclusion bodies; only a portion of them are expressed as soluble forms; and a protein fused with a target protein functions to aid the folding of the target protein (Savvas C. Makrides (1996), Microbiological Review, 512-538).
Thus, there is an urgent need for techniques allowing the high level production of biologically-active, soluble recombinant proteins in high efficiency and high concentrations.